Normozoospermic Men Research Articles

Beneficial Role of Platelet –Rich Plasma (PRP) combined with Glass Wool Filtration for Best Recovery of Spermatozoa in Infertile Men

Background: Platelet –Rich Plasma (PRP) is a novel therapeutic agent used in multiple medical fields and one of these fields is the reproduction for best spermatozoa preparation and activation for upgrading activity and motility of the spermatozoa and filtered with glass wool filtration to eliminate any round cells and leukocytes from the sample.
 Objectives: The aim of this research is to study some sperm characteristics in two groups, the normozoospermic infertile men and th asthenozoospermic infertile group before and after activation with Glass wool Filtration and PRP and compare between both.
 Patients and Methods: In this study 60 infertile men were enrolled and divided into 2 groups,15 normozoospermic infertile men, and 45 ashenozoospermic infertile men during their attendance to the Infertility Clinic in High Institute for Infertility Diagnosis and Assisted Reproductive Technologies Al-Nahrain University. The collected semen samples, and seminal fluid analysis were assessed, each semen sample was divided into 3 tubes and labelled as the 1st tube which was before activation, the 2nd tube was after glass wool filtration activation technique and the 3rd tube was for semen activated by adding PRP to the medium used for glass wool filtration activation.
 Results: Both techniques showed increased and improved sperm motility, but the PRP was superior to the glass wool alone in upgrading Sperm Grade A Motility %, Sperm Grade B Motility % and decreasing Sperm Grade C Motility %, and Sperm Grade D Motility (Immotile Sperm).
 Conclusion: The PRP was significantly effective in improving the sperm activity and upgrading sperm motility more than Glass Wool Filtration technique.

Does a Very Short Length of Abstinence Improve Assisted Reproductive Technique Outcomes in Infertile Patients with Severe Oligo-Asthenozoospermia?

In recent years, a growing number of studies seem to support the beneficial effects of a very short abstinence period on sperm parameters, especially in patients with oligo-asthenozoospermia (OA). On this basis, the aim of this study was to evaluate the effects of a short period of abstinence (1 h) on intracytoplasmic sperm injection (ICSI) outcomes in infertile patients with severe OA. We performed a retrospective study on 313 ICSI cycles in which couples were divided into two different groups based on sperm parameters of the male partners. Group 1 included normozoospermic men or male partners with a mild OA (n = 223). Group 2 included male partners with severe OA (n = 90). They were asked to provide a second consecutive ejaculation after 1 h from the first one. The best ejaculate was used to perform ICSI. We found a significant increase of total (p < 0.001) and progressive motility (p < 0.001) in the second ejaculate of patients of Group 2 compared with those of the first one. Spermatozoa of the second ejaculate were chosen for ICSI for all patients in Group 2. We found statistically significant improvement of clinical pregnancy rate (p = 0.001) and embryo quality (p = 0.003) in couples in Group 2 compared to those of Group 1. No statistically significant difference was found in fertilization, implantation, live birth delivery, and miscarriage rates between the two groups. Therefore, a second semen sample collected after a very short time-interval in patients with severe OA allowed us to obtain significantly higher clinical pregnancy rate with improved embryo quality compared to normozoospermic men or patients with mild OA. Fertilization, implantation, live birth delivery, and miscarriage rates were similar between the two groups. The present study shows that a second consecutive ejaculate could represent a simple strategy to obtain better sperm parameters and assisted reproductive technology (ART) outcomes in infertile patients with mild-severe OA.

The Impact of Luteinizing Hormone/Chorionic Gonadotropin Hormone Receptor Gene Polymorphism rs68073206 in Men with Non-obstructive Azoospermia: A Case-control Study

Background: The functional consequences of the luteinizing hormone/chorionic gonadotropin hormone receptor (LHCGR) gene single nucleotide polymorphism (rs68073206) on male infertility in patients with non-obstructive azoospermia (NOA) is not clear.&#x0D; Objective: To examine whether the presence of LHCGR gene; rs68073206 single nucleotide polymorphisms (SNPs) can be associated with incidence of non-obstructive azoospermia.&#x0D; Materials and methods: A case-control study comprised of a total of 70 unrelated Iraqi infertile men with non-obstructive azoospermia (zero sperm in semen) whose were on two groups: Group I that were diagnosed to have NOA but didn’t receive infertility treatment yet (33 patient with age of 31.58±1.059 year) and group II that were receiving injectable gonadotropin treatment (37 patient with age of 33.46±1.173 year). In addition to 34 age and BMI matched healthy fertile normozoospermic men (according to the parameters of WHO, 2010). The study population was genotyped by TaqMan assay for LHCGR gene single nucleotide polymorphism (rs68073206). The level of each hormone was estimated by immunoassay technique while the sperm analyses were conducted in accordance with the World Health Organization criteria.&#x0D; Results: The study revealed a statistically significant higher hormonal level of serum inhibin B in infertile group I patients with wild GG genotype (246.445±224.106 pg/ml), and the p-value is (0.0439) as compared to that hormone levels of GT and TT genotypes carriers that were (85.969±71.685 pg/ml) and (56.420±23.988 pg/ml) respectively. ). The genotyping variations of patients, whether carrying the homozygous GG, heterozygous GT or homozygous TT genotype, did not reveal a statistically significant difference in distribution as compared to control individuals.&#x0D; Conclusions: The LHCGR gene rs68073206 polymorphisms in our population having non-obstructive azoospermia can be suggested to have a modulating potential in variable gonadotropin sensitivity. The detected non-significant difference in genotypic prevalence can be attributable to the limited sample size.

Assembly of tetraspanins, galectin-3, and distinct N-glycans defines the solubilization signature of seminal prostasomes from normozoospermic and oligozoospermic men.

BackgroundProstasomes, extracellular vesicles (EVs) abundantly present in seminal plasma, express distinct tetraspanins (TS) and galectin-3 (gal-3), which are supposed to shape their surface by an assembly of different molecular complexes. In this study, detergent-sensitivity patterns of membrane-associated prostasomal proteins were determined aiming at the solubilization signature as an intrinsic multimolecular marker and a new parameter suitable as a reference for the comparison of EVs populations in health and disease.MethodsProstasomes were disrupted by Triton X-100 and analyzed by gel filtration under conditions that maintained complete solubilization. Redistribution of TS (CD63, CD9, and CD81), gal-3, gamma-glutamyltransferase (GGT), and distinct N-glycans was monitored using solid-phase lectin-binding assays, transmission electron microscopy, electrophoresis, and lectin blot.ResultsComparative data on prostasomes under normal physiology and conditions of low sperm count revealed similarity regarding the redistribution of distinct N-glycans and GGT, all presumed to be mainly part of the vesicle coat. In contrast to this, a greater difference was found in the redistribution of integral membrane proteins, exemplified by TS and gal-3. Accordingly, they were grouped into two molecular patterns mainly consisting of overlapped CD9/gal-3/wheat germ agglutinin-reactive glycoproteins and CD63/GGT/concanavalin A-reactive glycoproteins.ConclusionsSolubilization signature can be considered as an all-inclusive distinction factor regarding the surface properties of a particular vesicle since it reflects the status of the parent cell and the extracellular environment, both of which contribute to the composition of spatial membrane arrangements.

The Effect of Glyphosate on Human Sperm: In Vitro Approximation

Abstract Introduction Glyphosate is an herbicide used to eradicate illicit crops; however, its use is controversial due to different health problems associated with it. The present study aims to evaluate the effects of glyphosate on human sperm in vitro. Methods Twenty-two semen samples from healthy normozoospermic men were included; 11 semen samples were incubated with Panzer (INVESA S.A., Antiquia, Colombia) and 11 with Roundup (Monsanto Company, MO, USA). The changes in motility and viability were observed. Functional seminal parameters were evaluated as well. Results The samples exposed to glyphosate showed less motility and viability; a decrease in the potential of the mitochondrial membrane was observed, and an increase in the lipoperoxidation of the membrane was evidenced. Conclusion Based on the present results, we concluded that glyphosate has cytotoxic potential for exposed people and may affect their fertility.

CAG polymorphism of the Androgen Receptor gene and semen parameters in pathozoospermic patients with and without Y chromosome microdeletions, and in normozoospermic men

Introduction. The effect of polymorphic variants of the androgen receptor gene (AR) on spermatogenesis and semen parameters in men with different genotypes for other loci has not been sufficiently studied.The aim of this work was to study the effect of the (CAG)n polymorphism of the AR gene on semen parameters in men with impaired fertility, with and without partial deletions of the AZFс region from the Y chromosome.Materials and methods. The study included 988 unrelated Russian patients with pathozoospermia, including 591 patients without Y chromosome microdeletions and 397 patients with partial deletions of the AZFc region of the Y chromosome. The control group consisted of 131 normozoospermic men. All men who participated in the study underwent semen analysis and genetic testing. Genomic DNA was isolated from peripheral venous blood lymphocytes and ejaculate. The analysis of the polymorphism of (GAG)n repeat in exon 1 of the AR gene was performed using a polymerase chain reaction by the amplified fragment length polymorphism method.Results. Three groups were studied: patients with pathozoospermia with (n = 32) and without (n = 541) Y chromosome microdeletions, and normozoospermic men (control, n = 131). The median and quartiles of the number of CAG repeats in the groups were 22 and 20-25, respectively. According to the number of trinucleotide repeats of the AR gene, all patients were divided into subgroups: carriers of short ((GAG)n ≤18), medium ((GAG)n = 19-25) and long ((GAG)n ≥26) alleles. Medium alleles prevailed in all groups; in men without AZFc deletions and with microdeletions, their frequency was 79.3 and 81.4 %, respectively, in controls - 81.7 %.Conclusion. No correlation was found in examined cohort for semen parameters (sperm concentration and total number, number of live, progressively motile and morphologically normal spermatozoa) from the number of trinucleotide repeats. However, a statistically significant difference (p ≤0.045; FDR correction) was found in concentration and total number, number of live, progressively motile and morphologically normal spermatozoa when comparing men with nomrozoospermia (control) with patients with pathozoospermia with and without partial AZFc deletions in subgroups of carriers of short, medium and long alleles.

Sperm morphology and DNA fragmentation after zona pellucida selection.

Sperm DNA fragmentation (SDF) and sperm morphological defects can negatively affect ART outcomes. Consequently, there is a need for additional semen processing technique that accounts for sperm DNA status and morphology prior to ICSI. The objective was to evaluate the efficacy of an additional zona pellucida adhesion-based sperm selection for obtaining sperm populations with a high percentage of normal morphology and DNA integrity as compared to native semen and routine swim-up preparation. Semen samples from 78 normozoospermic men were subjected to swim up and placed in petri dishes coated with 48 acid-solubilized zonae pellucidae. Sperm DNA fragmentation and morphology were assessed in the native semen, the swim-up samples, and the zona-adhered spermatozoa from each patient. The mean sperm DNA fragmentation of the zona-selected spermatozoa (3.5 ± 0.7%) was significantly lower than the swim-up samples (15.3 ± 5.2%) (P < 0.001) and native semen (24.9 ± 7.1%) (P < 0.001). All of the samples had lower levels of DNA damage after additional selection by zona pellucida adhesion. Significantly higher percentage of sperm with normal morphology was observed after zona-adhesion selection (11.4 ± 3.9%) when compared to the swim-up samples (8.9 ± 4.3%) (P < 0.001) or the native semen (5.3 ± 3.2%) (P < 0.001). In 94% of the samples, the percentage of spermatozoa with normal morphology increased after the additional zona selection. This study demonstrates that sperm selection by additional zona-adhesion technique yields a significantly higher percentage of spermatozoa with normal morphology as well as a significantly decreased level of DNA fragmentation when compared to the native semen and the swim-up-only prepared samples.Lay summaryHigh level of DNA folding known as sperm DNA fragmentation (SDF) inside each sperm and defects in the shape, size, and structure of the sperm can negatively affect assisted reproduction treatment (ART) outcomes. Consequently, there is a need for additional semen processing techniques that account for sperm quality prior to ART. Our team designed a simple technique using proteins from the coat around the egg (zona pellucida) to enhance sperm selection procedures based on natural sperm–egg interactions. Using this technique in combination with the most common techniques used in ART yields a significantly higher percentage of sperm with normal shape, size, and structure and a decreased level of DNA fragmentation. This sperm zona-selection technique would be beneficial if introduced in the ART practice to yield sperm with higher fertilization potential.

Sperm DNA Damage and Protamine Transcripts Content in Iranian Men with Unexplained Infertility

Background: Incomplete chromatin condensation caused by altered amount of sperm protamines results in DNA fragmentation, which in turn leads to a lack of success in the development of the human embryo. Objectives: This study evaluated the sperm DNA damage and protamine transcripts content in Iranian normozoospermic fertile and infertile men. Methods: DNA damage was analyzed using comet assay. Transcript levels of protamine-1 (PRM1) and protamine-2 (PRM2) in ejaculated spermatozoa were assessed by quantitative real-time polymerase chain reaction (PCR). Results: Significantly higher levels of DNA damage were observed in unexplained infertile men (P = 0.001). DNA fragmentation correlated significantly with sperm total motility (r = -0.413, P = 0.032) and normal morphology (r = -0.424, P = 0.028). PRM1 and PRM2 transcripts contents were significantly lower in normozoospermic infertile men than healthy controls. Sperm PRM1 and PRM2 mRNA ratios were significantly higher (P = 0.035) in unexplained infertile patients than fertile men. Higher DNA damage was found to be significantly associated with reduced transcript levels of PRM1 (r = -0.453, P = 0.018) and PRM2 (r = -0.492, P = 0.009). Protamine transcripts ratios were significantly correlated with sperm normal morphology (r = -0.421, P = 0.029). Conclusions: Our findings showed the prognostic value and clinical utility of the sperm DNA damage and protamine transcripts contents for the discrimination between healthy fertile and unexplained infertile men.

O-147 Differential seminal metabolomic signature is related to sperm quality

Abstract Study question What is the entire metabolomic profile of human semen and does the metabolic composition differ between men with good-quality and low-quality semen? Summary answer Human semen contains ∼700 different metabolites, and the metabolomic signature differs between normozoospermic men and men with altered seminal parameters. What is known already Semen contains a wide diversity of metabolites as has been identified in single and targeted metabolite studies. The full composition of metabolites in human semen, however, is not known. The knowledge of the complete metabolic signature in semen and whether there are differences between metabolic composition and seminal quality could enhance our knowledge of possible factors involved in reduced sperm quality and male infertility. Study design, size, duration Case-control study, where a total of 100 men (age= 29.73±8.9 years) from March 2019 to March 2020 participated. The study was approved by the Ethics Committee of Investigación Biomédica de Andalucia. Participants/materials, setting, methods Semen samples from 69 normozoospermic and 31 oligozoospermic men were collected at the University Hospital and sperm biobank (Ceifer Biobank - NextClinics). The complete metabolome from unprocessed seminal samples was analysed by Ultrahigh Performance Liquid Chromatography-Tandem Mass Spectroscopy (UPLC-MS/MS). Raw data were extracted, peak-identified and quality control processed using Metabolon’s hardware and software (metabolon.com). Multiple regression models controlling for age and sample collection centres were applied using R software. Main results and the role of chance In total, 695 different metabolites were detected in the seminal samples, where docosahexaenoate (DHA; 22:6n3, PubChem ID 445580), choline phosphate (1014), dihomo-linolenate (20:3n3 or n6, 5280581), docosapentaenoate (n6 DPA; 22:5n6, 6441454), adenosine 3’,5’-cyclic monophosphate (cAMP, 6076) and N-acetylalliin (122164824) metabolites were the most prevalent. The seminal metabolomic profiles differed significantly between men with normal and low sperm parameters. The most abundant metabolites in normozoospermic men belonged to Lipid Super-Pathway, while Nucleotide Super-Pathway was predominant in semen samples with low quality (p &amp;lt; 0.05). More specifically, the leading Sub-Pathway in normozoospermic men was Long Chain Polyunsaturated Fatty Acid (n3 and n6), whereas Purine and Pyrimidine Metabolism Sub-Pathway prevailed in low-quality semen samples, where DHA and cAMP dominated in men with normal and low seminal quality parameters, respectively (p &amp;lt; 0.05 in all comparisons). Limitations, reasons for caution This is the first study presenting the entire metabolome signature of unprocessed human semen, and these preliminary results need to be confirmed in a bigger sample size. Wider implications of the findings Semen analyses applied in clinics do not evaluate the functional status of sperm, leaving the infertility causes due to male factor frequently unknown. Our study results could help to understand the molecular background of reduced seminal quality and male infertility and lead to identification of molecular biomarkers of functional sperm. Trial registration number not applicable

P–053 Concentration of bisphenol S in human seminal plasma negatively correlates with decreasing sperm concentration in ejaculate

Abstract Study question Is there a relationship between concentration of bisphenol S in seminal fluid and spermiogram parameters? Summary answer Bisphenol S was detected in 81% of seminal plasma samples. Negative correlation was found between BPS concentration and total sperm count in normozoospermic men. What is known already Human spermatogenesis can be influenced by a range of chemicals present in our environment. Bisphenol S (BPS) is a very frequent compound commonly used as a softener in production of plastics, where it has replaced bisphenol A. It is an endocrine disruptor frequently associated with negative effects on reproduction. It has been observed that BPS can affect testicular development in rodent males. In addition, it has cytotoxic, reprotoxic and neurotoxic effects and induces the oxidative stress bringing negative effects on spermatogenesis. BPS has been detected in food, drinks or cosmetics. Its direct effect on spermatozoa or spermatogenesis is still unclear. Study design, size, duration A total of 38 patients (25 normozoospermic) aged 24 to 42 years, non-smokers, with BMI between 19,9–32,9 were included in this prospective study from 2018 to 2020. None of them had varicocele, urogenital infections or other urological problems. Their seminal plasma was separated by centrifugation. BPS was extracted using solvent extraction followed by preconcentration step. The samples were analysed on Agilent 6495 Triple Quadrupole. Two MS/MS transitions were used for quantitative LC-MS/MS analyses. Participants/materials, setting, methods All the men included in this study signed an informed consent and agreed with analyses of their samples. These analyses were approved by Ethical committee of University Hospital Brno. We evaluated the relationship between concentration of BPS in seminal plasma, sperm concentration, total sperm count, total motility, progressive motility, morphology and fragmentation of DNA in spermatozoa. Statistical evaluation was performed by one individual one-dimensional regression model (p-value lower than 0.05 were considered as statistically significant). Main results and the role of chance The examination revealed the presence of BPS in 31 samples of seminal plasma (81% of all the samples). In 6 samples, the concentration was under level of detection and in one sample under level of quantification. In 7 samples, a very high concentration was detected (&amp;gt;0.1 ng/ml). These values were then compared to spermiogram parameters and sperm DNA integrity. There were no significant differences between the concentration of BPS and morphology of spermatozoa, progressive motility and total motility. In case of the DNA integrity, the opposite trend was observed, lower proportions of spermatozoa with fragmented DNA were found in samples with higher concentrations of BPS. Evaluation of sperm concentration and BPS concentration showed relationship of increasing BPS concentration with significantly lower sperm concentration, the differences were most obvious when only the normozoospermic men were compared. Evaluation of BPS concentration and total sperm count revealed the same trend with statistically significant difference in the category of normozoospermic men. Due to the small number of samples, a negative effect of extreme values on the statistical evaluation cannot be excluded. Currently, more analyses focused on detection of BPS in seminal plasma are carried out in order to obtain sufficiently larger data set. Limitations, reasons for caution A limitation is the number of samples included and analysed in this study, which slightly reduced the power of statistical analysis. Wider implications of the findings: These results document that BPS was present in 81% of analysed samples. Knowing the concentration of BPS in seminal fluid is important for understandig of impact of BPS on male fertility. Our future work will be focused on detection of other bisphenols in seminal plasma. Trial registration number MH CZ – DRO (FNBr, 65269705), AZV NV18–01–00544, Czech Ministry of Education, Youth and Sports (CZ.02.2.69/0.0/0.0/19_074/0012727)

The possible association of clusterin fucosylation changes with male fertility disorders

In the seminal plasma (n = 118) and serum (n = 90) clusterin (CLU) the fucosylation and the expression of selected fucosyltransferases (FUTs) were analyzed. Samples from infertile men were divided into groups based on the results of the standard semen analysis: normozoospermic (N), teratozoospermic (T), asthenoteratozoospermic (AT) and oligoasthenoteratozoospermic (OAT). The CLU fucosylation was analyzed using lectin-ELISAs with biotinylated lectins specific to α1,3-, α1,2-linked antennary fucose, and α1,6-linked core fucose (LTA, UEA, and LCA, respectively). The concentrations of FUT3 and FUT4, reflecting the expression of Le oligosaccharide structures, were measured using ELISA tests. The differences in serum CLU and FUT4 concentrations, and in the expression of core fucose and antennary fucose α1,2-linked in CLU glycans between the N group and other groups examined suggest that the disturbances in sperm count, motility, and morphology are not the only cause of male infertility. Lack of similarities between levels of examined parameters in blood serum and seminal plasma may suggest the differences in mechanisms leading to glycoproteins glycosylation. It confirmed the observed differences in concentrations of seminal plasma CLU, FUT3, and FUT4 between the OAT group and N, T, AT groups, indicating that decreased sperm count may be related to these parameters expression. The serum CLU concentrations and expression of core fucose and fucose α1,2-linked in CLU, seem to be good markers differentiating normozoospermic men from those with abnormal sperm parameters, which was not observed for seminal plasma.

Association of decreased sperm motility and increased seminal plasma IGF-I, IGF-II, IGFBP-2, and PSA levels in infertile men.

Previous studies have suggested the involvement of serum insulin-like growth factors (IGFs) and IGF binding proteins (IGFBPs) in the regulation of the female reproductive system. Little is known of these peptides in the seminal plasma (SP) of men and their potential effects on fertility. We assessed SP levels of these peptides in infertile men with low sperm motility (asthenozoospermic; AZ) and low sperm counts (oligozoospermic; OZ), its effects on in vivo sperm motility, and whether there is a correlation with aging. Twenty-eight infertile men (AZ; n = 18 and OZ; n = 10) and 20 fertile normozoospermic (NZ) men were studied. Seminal plasma IGF-I, IGF-II, IGFBP-2, IGFBP-3, and prostate-specific antigen (PSA) levels were measured, and spermatozoa mRNA transcript patterns were examined. Asthenozoospermic men had higher SP IGF-I, IGF-II, IGFBP-2, and PSA levels than NZ and OZ men, whereas SP IGFBP-3 levels were similar between the three groups. Sperm count positively correlated with SP IGF-I, IGF-II, and IGFBP-2; sperm motility negatively correlated with SP IGF-II and IGFBP-2; and age correlated positively with SP IGF-II. The expression of IGF-I and IGF-II mRNA and mRNA receptors was detectable, but no variations in transcript levels were noted. Decreased sperm motility, but not sperm count, in infertile AZ men is associated with increased SP IGF-I, IGF-II, IGFBP-2, and PSA levels. Changes in SP IGFs and their interactions with IGFBPs and IGF receptors, and PSA levels suggest a role of these SP peptides in modulating sperm motility and possibly prostate disease development in aging men.

Seminal plasma metabolomics profiles following long (4–7 days) and short (2 h) sexual abstinence periods

ObjectiveMetabolomic profiling of seminal plasma has been suggested as a possible approach for a fast and non-invasive male infertility evaluation diagnosis. However, metabolomics profiles in normozoospermic men have not been thoroughly investigated, and the influence of ejaculation-abstinence has not been described.To provide interim reference values and find associations between the metabolomics profiles of human seminal plasma and length of ejaculation-abstinence period in normozoospermic men. Study designSemen samples collected after long (4–7 days) and short abstinence (2 h) from 31 normozoospermic males were assessed for routine quality parameters before the seminal plasma was separated by centrifugation. Metabolomics profiles of the seminal plasma were then determined using untargeted Nuclear Magnetic Resonance Spectroscopy. ResultsIn total, 30 metabolites were identified. Pyruvate showed a higher concentration, while fructose, acetate, choline, methanol, N-acetylglucosamine, O-acetylcarnitine, uridine, and sn-glycero-3-phosphocoline showed lower concentrations in samples collected after short abstinence (vs. long). All metabolites showed lower absolute amounts (volume × concentration) following shorter abstinence. However, the lower sperm concentration in samples collected after short abstinence resulted in higher absolute amounts of pyruvate and taurine per spermatozoa: pyruvate 1.92 (1.12–3.87) vs. 1.29 (0.83–2.62) (P < 0.001) and taurine 0.58 (0.36–0.92) vs. 0.43 (0.28–0.95) (P < 0.05) ng/106 spermatozoa. Simultaneously, there was a higher percentage of progressively motile spermatozoa in samples collected after the short abstinence. ConclusionThe generally lower concentrations of seminal metabolites after short abstinence periods may be related to the shorter time available for secretion and collection of these metabolites by the accessory glands and the epididymides. The concomitant lower number of spermatozoa in the second ejaculate resulted in increased absolute amounts of pyruvate and taurine per spermatozoa, accompanied by increased spermatozoa motility in these samples.The simultaneous increase in percentages of motile spermatozoa and absolute amounts of pyruvate and taurine per spermatozoa after shorter abstinence might indicate that these two metabolites play a more critical role in sperm motility, which should be further investigated in future studies.

Decreased piRNAs in Infertile Semen Are Related to Downregulation of Sperm MitoPLD Expression.

Currently, the molecular mechanisms underlining male infertility are still poorly understood. Our previous study has demonstrated that PIWI-interacting RNAs (piRNAs) are downregulated in seminal plasma of infertile patients and can serve as molecular biomarkers for male infertility. However, the source and mechanism for the dysregulation of piRNAs remain obscure. In this study, we found that exosomes are present in high concentrations in human seminal plasma and confirmed that piRNAs are predominantly present in the exosomal fraction of seminal plasma. Moreover, we showed that piRNAs were significantly decreased in exosomes of asthenozoospermia patients compared with normozoospermic men. By systematically screening piRNA profiles in sperms of normozoospermic men and asthenozoospermia patients, we found that piRNAs were parallelly reduced during infertility. At last, we investigated the expression of some proteins that are essential for piRNAs biogenesis in sperms and therefore identified a tight correlation between the levels of spermatozoa piRNA and MitoPLD protein, suggesting that the loss-of-function of MitoPLD could cause a severe defect of piRNA accumulation in sperms. In summary, this study identified a parallel reduction of piRNAs and MitoPLD protein in sperms of asthenozoospermia patients, which may provide pathophysiological clues about sperm motility.

Proposed age-stratified reference intervals of FSH derived from normozoospermic men

Objective: To demonstrate that serum follicle-stimulating hormone (FSH) in men rises with age, and to explore FSH reference intervals of age-related partitioning. Methods: Men aged 20-50 years (n=1 190) underwent semen analysis according to World Health Organization (2010) methods. Serum was frozen prior to measurement of FSH by using the Siemens ADVIA Centaur® XP immunoassay system. FSH central 95% intervals after logarithmic transformation based on age were derived from 1 037 normozoospermic men. These were then applied to oligozoospermic and azoospermic men. Men producing azoospermic semen samples were further classified as having non-obstructive azoospermia by clinical diagnostic criteria, including genetic analysis and surgical exploration. Results: Serum FSH in normozoospermic men increased with age (P&lt;0.05), and reference intervals were determined with 10-year brackets: 21-30 years [(1.0-8.2) IU/L], 31-40 years [(1.4-9.5) IU/L], 41-50 years [(1.9-12.0) IU/L]. The proportion of oligozoospermic men with normal FSH concentrations was less than the normozoospermic men, which in turn was lower among azoospermic men (both P&lt;0.01). The azoospermic men were further broken down according to the nature of the azoospermia as either obstructive or non-obstructive azoospermia, and 86.4% (38/44) men with non-obstructive azoospermia had elevated serum FSH concentrations whereas only 6.7% (1/15) men with obstructive azoospermia had high FSH levels, and this was significantly different (P&lt;0.01). Conclusions: FSH concentrations increase in men between 20-50 years, and clinical interpretation of serum FSH results in men must be made by using age-based reference intervals.

Study the effect of mixing platelet activating factor with sperm preparation medium of normozoospermic men on intra uterine insemination outcome

Intrauterine insemination (IUI), it is a type of artificial insemination, used to treat infertility by which the Sperm that have been prepared and placed directly into the woman’s uterus around the time of ovulation. The main purpose of this procedure is to enable the sperm to swim into the fallopian tubes and fertilize a ready egg in order to lead to a natural pregnancy. IUI can be coordinated with the normal cycle with fertility or medications depending on the reasons for infertility The IUI outcome still low about 20-25% so adding number of biochemical factors to the culture medium which are essential to regulate the fertility potential of spermatozoa, for example, platelet-activating factor (PAF) which is a unique and novel signaling phospholipid that has multiple biologic properties have been tried. To determine the effect of adding the PAF into the culture medium to prepare and activate the semen samples of non-male factor infertility (normal semen analyses). This study was carried out in the Infertility Unit, AL-Hussein Teaching Hospital, Al-Nassirya City, through the period from 30th of October 2020 till 17th of April 2021. The study included thirty couples who are complaining from infertility (they failed to conceive after one year at least of regular unprotected intercourse. The frequency distribution of enrolled infertile men with normozoospermia Baseline characteristics of seminal fluid analysis of males with normozoospermia those with PAF are Semen volume ml, pH, Sperm concentration before million/ml, Sperm motility %, morphologically normal sperm % , were Mean ±SD (3.10 ±1.23, 7.39 ±0.03, 46.67 ±15.31, 1.33 ±3.52, 36.00 ±5.07) respectively. Certain sperm parameters were highly improved after in vitro activation by PAF medium of normozoospermic samples.

Study the effect of mixing platelet activating factor with sperm preparation medium of normozoospermic men on intra uterine insemination outcome

Intrauterine insemination (IUI), it is a type of artificial insemination, used to treat infertility by which the Sperm that have been prepared and placed directly into the woman’s uterus around the time of ovulation. The main purpose of this procedure is to enable the sperm to swim into the fallopian tubes and fertilize a ready egg in order to lead to a natural pregnancy. IUI can be coordinated with the normal cycle with fertility or medications depending on the reasons for infertility The IUI outcome still low about 20-25% so adding number of biochemical factors to the culture medium which are essential to regulate the fertility potential of spermatozoa, for example, platelet-activating factor (PAF) which is a unique and novel signaling phospholipid that has multiple biologic properties have been tried. To determine the effect of adding the PAF into the culture medium to prepare and activate the semen samples of non-male factor infertility (normal semen analyses). This study was carried out in the Infertility Unit, AL-Hussein Teaching Hospital, Al-Nassirya City, through the period from 30th of October 2020 till 17th of April 2021. The study included thirty couples who are complaining from infertility (they failed to conceive after one year at least of regular unprotected intercourse. The frequency distribution of enrolled infertile men with normozoospermia Baseline characteristics of seminal fluid analysis of males with normozoospermia those with PAF are Semen volume ml, pH, Sperm concentration before million/ml, Sperm motility %, morphologically normal sperm % , were Mean ±SD (3.10 ±1.23, 7.39 ±0.03, 46.67 ±15.31, 1.33 ±3.52, 36.00 ±5.07) respectively. Certain sperm parameters were highly improved after in vitro activation by PAF medium of normozoospermic samples.

Immunological response of fallopian tube epithelial cells to spermatozoa through modulating cytokines and chemokines

BackgroundSpermatozoa interactions with fallopian tubes may influence fertilization. The purpose was to investigate cytokines, chemokines and growth factors expression from human fallopian tube epithelial cells (OE-E6/E7) exposed to spermatozoa. MethodsFresh semen samples were obtained from 10 healthy normozoospermic men. Sperms were prepared and co-cultured with OE-E6/E7. The cell line without spermatozoa was considered as the control group. Afterwards, Expression of 84 cytokines from OE-E6/E7 cell line in the presence and absence of spermatozoa were measured using PCR-array. Quantitative PCR was performed on seven genes to confirm the results of PCR-array analysis. Differentially expressed genes were subjected to www.geneontology.org and www.pantherdb.org to perform GO enrichment and panther pathway analysis. The concentration of IL-8, IL-10, IL-1B and BMP-4 in culture medium were analyzed by ELISA. ResultsSperm interaction with the epithelial cells resulted in a significant increase in expression of TGF-β2, BMP-4, IL-10, IL-9, and CD40LG markers. Moreover, expression of IL-16, IL-17F, SPP-1, CXCL-13, MSTN, IL-1A, IL-1B, IL-8, BMP-7, CSF-2, CSF-3, VEGF-A, OSM, LTA, TNF, TNFRSF11B, TNFSF11, CCL-11, CCL-20, CCL-24, CCL-3, CCL-8, CX3CL1 and CXCL-9 were considerably reduced in presence of spermatozoa. Panther pathway analysis discovered 3 pathways for upregulated genes including gonadotropin-releasing hormone receptor, TGF-beta and interleukin signaling pathways. Furthermore, 9 pathways were detected for down-regulated genes. Inflammation signaling pathway which is mediated by chemokine and cytokine contains the most number of genes. ConclusionThis study indicates that sperm modifies expression of cytokines, chemokines and growth factors from OE-E6/E7. Moreover, altered genes expression are toward higher survival chance of the spermatozoa.

Association of Vitamin D and Reproductive Hormones With Semen Parameters in Infertile Men.

BackgroundHypovitaminosis D has been linked with poor semen parameters and endocrinological factors in male infertility. This study aimed to analyze the association of serum vitamin D levels and reproductive hormones such as follicle-stimulating hormone (FSH), luteinizing hormone (LH), and total testosterone (TT) with the semen parameters in a cohort of infertile men.MethodologyIn this study, 224 infertile men (aged 18-45 years) were recruited after applying inclusion and exclusion criteria. Semen analysis was performed as per the 2010 World Health Organization (WHO) criteria. The patients were divided into two groups on the basis of semen parameters: normozoospermic men and men with one or more altered semen parameters as per the WHO 2010 guidelines for all the analysis. Vitamin D and hormone levels were evaluated by quantitative competitive immunoassay by chemiluminescent microparticle immunoassay technology with flexible assay protocols (Architect, Abbott Diagnostics, Lake Forest, IL, USA). The patients were further divided into three groups on the basis of vitamin D levels: Sufficient (>20 ng/mL), insufficient (12-20 ng/mL), and deficient (<12 ng/mL). These groups were compared for different semen and hormonal parameters.ResultsOut of the 224 infertile men included, 124 were normozoospermic while 100 patients had one or more altered semen parameters. The median age of the participants was 30 years (minimum = 18 years, maximum = 45 years). The serum vitamin D and TT levels were significantly lower (p < 0.0001) and FSH (p = 0.001) and LH levels (p < 0.0001) were significantly higher in those with one or more altered semen parameters compared to the normozoospermic men. The sperm concentration, total motility, linear progressive motility, percentage normal morphology, and serum TT levels were significantly lower in the patients with serum vitamin D levels of <12 ng/mL in both the normozoospermic men and those with one or more altered semen parameters compared to patients with higher vitamin D levels. Semen parameters such as sperm concentration, total motility, linear progressive motility, and morphology were positively correlated with the levels of serum vitamin D and TT.ConclusionsVitamin D deficiency was examined in a large proportion of infertile men. Serum vitamin D and TT levels were positively associated with semen parameters evident from lower levels of vitamin D and TT in men with altered semen parameters compared to normozoospermic men. However, further comprehensive studies with larger sample sizes should be conducted to further validate the role of vitamin D in male infertility by checking the effect of vitamin D supplementation on semen parameters.

Mutations in the desert hedgehog (DHH) gene in the disorders of sexual differentiation and male infertility.

To identify the contribution of mutations in the Desert Hedgehog (DHH) gene to the disorders of sexual differentiation (DSD) and male infertility. The study included a total 430 subjects, including 47 gonadal dysgenesis cases, 6 patients with undescended testis and infertility characterized by azoospermia, 125 infertile male patients characterized by oligoasthenozoospermia, 24 patients with oligoasthenoteratozoospermia, and 200 ethnically matched normozoospermic fertile men who had fathered a child in the last two years. Sequencing of the complete coding region of the DHH gene was undertaken to find its contribution to the DSD and male infertility. We observed four novel mutations in the DHH gene in the cases with different reproductive anomalies. A synonymous substitution, c. 543C>T (p.His181His) was observed in 6.6% oligoasthenozoospermic infertile males and 1.5% normozoospermic fertile control samples (RR = 4.4077, 95%CI 1.19-16.29). Another synonymous substitution, c.990G>A (p.Ala330Ala) was observed in an infertile patient with unilateral undescended testis (case #12). Insertion of G at c.1156insG (p.Arg385fs) was observed in a case with bilateral undescended testis and azoospermia (case #23). In gonadal dysgenesis category, two mutations, insertion of G at c.1156insG (p.Arg385fs) and c.997A>G (p.Thr333Ala) substitution were observed in one case (case #34). These mutations were completely absent in control samples. Mutations in the DHH gene impact reproduction with mild mutations affecting fertility, and severe or multiple mutations resulting in gonadal dysgenesis.